牦牛乳腺上皮细胞体外培养及鉴定

doi:10.11843/j.issn.0366-6964.2015.01.009

Abstract

Abstract:

The present study was carried out to establish yak mammary epithelial cell (YMEC) line and identify its biological characteristic，which was separated using tissue explants method，purified by selective trypsinization，characterized by immunofluorescence and Western blot assay.Transfecting EGFP-N1 gene into cells using lipofection to examine whether foreign genes could be transfected into YMECs，fluorescence microscope was used to detect the expression of EGFP gene.Lastly，flow cytometry was used to evaluate the apoptosis level of cells with Annexin V/PI double staining and RT-PCR was used to examine gene expression of antibacterial peptide and apoptosis factors to determine the effect of Staphylococcus aureus infection on YMECs.Immunofluorescence assay results proved established cell line could express cytokeratin 18 rather than vimentin，indicating no fibroblast existing in epithelial cells.Positive expression of β-casein in YMECs demonstrated cultured cells could synthesize and secrete mammary- specific milk protein.Moreover，we detected EGFP protein in transfected YMECs by fluorescence microscope，showing EGFP gene was successfully transfected into the YMECs.After YMECs were challenged with for Staphylococcus aureus for 3 h，the apoptosis level of mammary epithelial cells was significantly up-regulated（P<0.05）.Furthermore，the expression of TAP（P<0.01），BNBD5（P<0.01）and BAX（P<0.01）genes increased; whereas that of BCL-2 gene decreased（P<0.05）.To conclude，we successfully established a stable yak mammary epithelial cell line，which can be used as a useful model in vitro for study of yak mammary gland development and differentiation.

CLC Number:

CHEN Ya-bing，FU Mei，LAN Dao-liang，LIN Bao-shan，HUANG Cai，LI Jian. Culture and Identification of Yak Mammary Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(1): 69-76.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2015.01.009

https://www.xmsyxb.com/EN/Y2015/V46/I1/69

References

［1］SUN Y L，LIN C S，CHOU Y C.Establishment and characterization of a spontaneously immortalized porcine mammary epithelial cell line ［J］.Cell Biol Int，2006，30(12)：970-976.
［2］SHI H，LUO J，WANG W，et al.Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase(hT-MECs) ［J］.Anim Sci J，2014，85(7)：735-743.
［3］TONG H L，LI Q Z，GAO X J，et al.Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line ［J］.In vitro Cell Dev Biol Anim，2012，48(3)：149-155.
［4］ZHAO K，LIU H Y，ZHOU M M，et al.Establishment and characterization of a lactating bovine mammary epithelial cell model for the study of milk synthesis ［J］.Cell Biol Int，2010，34(7)：717-721.
［5］GAFFNEV E V，POLANOWSKI F，BLACKBURN S E，et al.Cultures of normal human mammary cells ［J］.Cell Differ，1976，5(2)：69-81.
［6］YANG J，RICHARDS J，GUZMAN R，et al.Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels ［J］.Proc Natl Acad Sci U S A，1980，77(4)：2088-2092.
［7］PANTSCHENKO A G，WOODCOCK-MITCHELL J，BUSHMICH S L，et al.Establishment and characterization of a caprine mammary epithelial cell line(CMEC) ［J］.In vitro Cell Dev Biol Anim，2000，36(1)：26-37.
［8］KE M W，HSU J T，JIANG Y N，et al.Characterization of hTERT-immortalized caprine mammary epithelial cells ［J］.Reprod Domest Anim，2012，47(4)：553-561.
［9］HU H，WANG J，BU D，et al.In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow ［J］.PLoS ONE，2009，4(11)：e7636.
［10］JEDRZEJCZAK M，SZATKOWSKA I.Bovine mammary epithelial cell cultures for the study of mammary gland functions ［J］.In vitro Cell Dev Biol Anim，2014，50：389-398.
［11］ANAND V，DOGRA N，SINGH S，et al.Establishment and characterization of a buffalo(Bubalus bubalis) mammary epithelial cell line ［J］.PLoS ONE，2012，7(7)：e40469.
［12］KAUSHIK R，SINGH K P，KUMARI A，et al.Isolation，characterization，and EGFP expression in the buffalo(Bubalus bubalis) mammary gland epithelial cell line［J］.In vitro Cell Dev Biol Anim，2013，49(1)：1-7.
［13］WHEELER T T，CALLAGHAN M R，DAVIS S R，et al.Milk protein synthesis，gene expression，and hormonal responsiveness in primary cultures of mammary cells from lactating sheep ［J］.Exp Cell Res，1995，217(2)：346-354.
［14］王桢，罗军，王伟，等.奶山羊乳腺上皮细胞的分离、培养及鉴定［J］.生物工程学报，2010，26(8)：1123-1127.
WANG Z，LUO J，WANG W,et al.Characterization and culture of isolated primary dairy goat mammary gland epithelial cells［J］.Chinese Journal of Biotechnology，2010，26(8)：1123-1127.(in Chinese)
［15］陈建晖，佟慧丽，李庆章，等.奶牛乳腺上皮细胞系的建立［J］.畜牧兽医学报，2009，40(5)：743-747.
CHEN J H,TONG H L,LI Q Z,et al.Establishment of dairy cow mammary gland epithelial cell line ［J］.Acta Veterinaria et Zootechnica Sinica，2009，40(5)：743-747.(in Chinese)
［16］AKERS R M.Major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows ［J］.J Dairy Sci，2006，89(4)：1222-1234.
［17］PUISSANT C，ATTAL J，HOUDEBINE L M.The hormonal control of ovine beta-lactoglobulin gene in cultured ewe mammary explants ［J］.Reprod Nutr Dev,1990,30(2)：245-251.
［18］WHEELER M B，WALTERS E M.Transgenic technology and applications in swine ［J］.Theriogenology，2001,56(8)：1345-1369.
［19］KUMURA H，TANAKA A，ABO Y，et al.Primary culture of porcine mammary epithelial cells as a model system for evaluation of milk protein expression ［J］.Biosci Biotechnol Biochem，2001，65(9)：2098-2101.
［20］YANG Z T，FU Y H，LIU B，et al.Farrerol regulates antimicrobial peptide expression and reduces Staphylococcus aureus internalization into bovine mammary epithelial cells［J］.Microb Pathog，2013，65:1-6.
［21］WEI Z K，FU Y H，ZHOU E S，et al.Effects of niacin on Staphylococcus aureus internalization into bovine mammary epithelial cells by modulating NF-kB activation［J］.Microb Pathog，2014,（71-72）：62-67.
［22］崔新洁，胡庆亮，李奕平，等.金黄色葡萄球菌诱导牛原代乳腺上皮细胞的凋亡［J］.中国农业科学，2013，46(15)：3212-3219.
CUI X J，HU Q L，LI Y P，et al.The apoptosis of bovine primary mammary epithelial cells induced by Staphylococcus aureus［J］.Scientia Agricultura Sinica，2013，46(15)：3212-3219.(in Chinese)
［23］HU Q，CUI X，TAO L，et al.Staphylococcus aureus induces apoptosis in primary bovine mammary epithelial cells through Fas-FADD death receptor-linked caspase-8 signaling［J］.DNA Cell Biol，2014，33(6)：388-397.

Culture and Identification of Yak Mammary Epithelial Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 0

Recommended Articles

Metrics

Comments